Clefts of the lip and/or palate (CL/P) are the most common human craniofacial birth defect (1/700 births). The mouse offers a suitable model to study human craniofacial morphogenesis and its abnormalities, which remain poorly understood. It was reported that a unique cellular behavior known as Epithelial Mesenchymal Transition (EMT) mediates craniofacial prominence fusion in the chick. However, the underlying mechanisms were not determined and it remains unknown whether EMT controls upper lip morphogenesis in mammals. Our evidence indicates that: 1) in the mouse embryo, in addition to apoptosis, epithelial plasticity at the prominence seam mediates tissue remodeling and fusion; 2) this process is dependent upon Pbx transcription factors (TFs); 3) epithelial cells at the Pbx mutant seam lose expression of Snail1 and nuclear Smad3/4, critical EMT mediators; and 4) Pbx TFs bind to Snail1 and Smad3 potential regulatory elements, as demonstrated by Chromatin immunoprecipitation (ChIP) on midface tissue. Moreover, whole-genome sequence analysis of Pbx1-bound regions in the murine face identifies additional potential Pbx target genes associated with epithelial plasticity, EMT, cell adhesion, and migration. In view of these results, we posit that, in addition to controlling apoptosis, Pbx TFs act as novel regulators of epithelial plasticity or EMT at the seam. We also posit that Pbx TFs concomitantly control closely related cellular behaviors, namely cell adhesion and migration, in mouse midface morphogenesis. First, we will determine whether local epithelial plasticity or complete EMT mediates tissue remodeling and prominence fusion at the seam. To this end, we will define the requirement for Pbx in the control of epithelial plasticity in the embryonic head using our Pbx-deficient mouse lines. Then we will investigate whether Pbx TFs are necessary and also sufficient to drive epithelial plasticity or complete EMT using NMuMG epithelial cells, a classic model of EMT. Second, we will establish whether Snail1 and Smad3 are regulated by Pbx TFs in midface morphogenesis. To achieve this, we will perform transient transfections in embryonic cells and transient transgenesis in the mouse. Third, we will delineate a comprehensive regulatory network of Pbx-directed effectors of epithelial plasticity, EMT, cell adhesion and migration in murine face morphogenesis. To this end, we will determine which Pbx1-bound enhancers identified by ChIP-Seq on midface tissue regulate Pbx target genes by transcriptome assays on wild type and Pbx-mutant cephalic epithelium. We will validate selected novel Pbx-regulated genes in vivo, with priority based on current findings for candidates Zeb1 & Zeb2 and Zpo1 & Zpo2, all of which are effectors of EMT, cell adhesion, or migration. This re- search will identify novel craniofacial cis-regulatory elements and Pbx-directed networks driving interconnected cellular behaviors that are critical for face morphogenesis, as well as revealing new Pbx target genes for pre- natal diagnostics of CL/P. Investigations on basic mechanisms underlying epithelial behaviors in head development will have applications to other processes that rely on the same cell behaviors, e.g., tumor invasion.